Adverse Metabolic Phenotypes in Parenterally Fed Neonatal Pigs Do Not Persist into Adolescence.

BACKGROUND: Nutrition during fetal and neonatal life is an important determinant for the risk of adult-onset diseases, especially type 2 diabetes and obesity. OBJECTIVES: We aimed to determine whether total parenteral nutrition (TPN) compared with enteral formula feeding [enteral nutrition (EN)] in term piglets during the first 2 wk after birth would increase the long-term (5-mo) development of metabolic syndrome phenotypes with adverse glucose homeostasis, fatty liver disease, and obesity. METHODS: Neonatal female pigs were administered TPN (n = 12) or fed enterally with a liquid enteral milk-replacer formula (EN, n = 12) for 14 d. After transitioning TPN pigs to enteral feeding of liquid formula (days 15-26), both groups were adapted to a solid high-fat diet (30% of the total diet) and sucrose (20% of the total diet) diet (days 27-33), which was fed until the end of the study (140 d). Body composition was measured by dual-energy X-ray absorptiometry at 14, 45, and 140 d. Serum biochemistry and glucose-insulin values (after a fasting intravenous glucose tolerance test) were obtained at 140 d. Liver and muscle were analyzed for insulin receptor signaling and triglycerides. RESULTS: Body weight was similar, but percent fat was higher, whereas percent lean and bone mineral density were lower in TPN than in EN pigs (P < 0.01) at 45 d of age but not at 140 d. At 140 d, there were no differences in serum markers of liver injury or lipidemia. Intravenous glucose tolerance test at 140 d showed a lower (P < 0.05) AUC for both glucose and insulin in TPN than in EN pigs, but the ratio of AUCs of insulin and glucose was not different between groups. CONCLUSIONS: Administration of TPN during the neonatal period increased adipose deposition that transiently persisted in early adolescence when challenged with a high-fat diet but was not sustained or manifested as glucose intolerance.

Enteral plasma feeding improves gut function and immunity in piglets after birth asphyxia.

INTRODUCTION: Birth asphyxia may negatively affect gut function and immunity in newborns. Conversely, immunomodulatory milk diets may protect the gut and immune system against damage caused by asphyxia. Using caesarean-derived pigs as models, we hypothesised that enteral feeding with plasma improves gut and immune functions in asphyxiated newborns. METHODS: Near-term pig fetuses (98% gestation,) were delivered by caesarean section after 8 min umbilical cord occlusion, leading to transient birth asphyxia (ASP, n = 75) and compared with non-occluded controls (CON, n = 69). Piglets were further randomised to supplementation with/without porcine plasma (plasma, PLA/vehicle, VEH), into bovine colostrum (first 24 h) or formula (until 72 h). RESULTS: Compared with CON, ASP piglets took longer to achieve stable respiration and showed reduced blood pH, weight gain and survival. Independent of asphyxia, plasma supplementation reduced gut haemorrhagic lesions, permeability and inflammatory cytokines together with improved villous morphology and brush-border enzyme activities. Asphyxia reduced blood cytokine responses to ex vivo bacterial stimulation, whereas plasma supplementation ameliorated this effect. CONCLUSION: Dietary plasma supplementation improves survival, gut functions and immunity in both normal and asphyxiated newborns. The components in plasma that mediate gut-protective effects in piglets remain to be identified, but may benefit also birth-compromised newborn infants. IMPACT: Complicated deliveries leading to birth asphyxia, may negatively affect gut, liver and immune adaptation in the first days after birth. Using a model of birth asphyxia in caesarean-derived piglets, we show that enteral feeding with maternal plasma exerts gut maturational and immunomodulatory effects in both control and asphyxiated animals in the first days of life. The mechanisms behind the gut-protective effects of plasma are unknown, but plasma components hold potential for new oral therapies for compromised newborn infants as well as piglets.

Trial-related blood sampling and red blood cell transfusions in preterm infants.

AIM: To determine if trial-related blood sampling increases the risk of later red blood cell (RBC) transfusion in very preterm infants, we compared the volume of clinical- and trial-related blood samples, in a specific trial and correlated to subsequent RBC transfusion. METHODS: For 193 very preterm infants, participating in the FortiColos trial (NCT03537365), trial-related blood volume drawn was in accordance with ethical considerations established by the European Commission. Medical records were reviewed to assess the number and accumulated volume (mL/kg) of blood samples (both clinical- and trial-related). Data were compared with the need of RBC transfusions during the first 28 days of life. RESULTS: Mean (SD) gestational age and birth weight was 28 ± 1 weeks and 1168 ± 301 g. In total, 11% of total blood volume was drawn for sampling (8.1 ± 5.1 mL/kg) and trial-related sampling accounted for 1.6 ± 0.6 mL/kg. Trial-related blood sampling had no impact on RBC transfusion (p = 0.9). CONCLUSION: Clinical blood sampling in very preterm infants is associated with blood loss and subsequent need for RBC transfusions. In a specific trial requiring blood samples, we found no additional burden of trial-related blood sampling. The study suggests that trial-related sampling is safe if European criteria are followed.

Nurses' and Physicians' Rationale Behind Clinical Performance and Interpretation of Routine Prefeed Gastric Aspiration in Preterm Infants: A Cross-sectional Study.

This study aims at understanding the rationale behind performing prefeed gastric aspirations in preterm infants, how nurses and physicians interpret the gastric aspiration and variations between them, and illuminating potential barriers for omitting routine prefeed aspiration. Nurses and physicians from all Danish neonatal intensive care units completed a questionnaire. Of 682 participants, the majority (94%) indicated that they routinely performed prefeed aspiration, primarily to check the feeding tube placement (nurses: 88%, physicians: 46%). Nurses feared necrotizing enterocolitis when observing a large gastric residual (GR) volume (31%) and green-stained GR (63%). Fewer nurses relative to physicians had "no worries" related to large volumes (15% vs 34%) or green-stained GR (14% vs 24%, both P < .01). More nurses than physicians intended to pause enteral feeding when observing green-stained GR (31% vs 16%, P < .01) and more nurses were concerned of completely omitting routine gastric aspirations (90% vs 46%, P < .05). The rationale behind the clinical use of GR volume and color as markers of necrotizing enterocolitis and feeding intolerance differs markedly between nurses and physicians in Denmark. If routine prefeed gastric aspiration should be omitted, special focus on information about early signs of necrotizing enterocolitis and methods to check tube placement is needed.

Subclinical necrotizing enterocolitis-induced systemic immune suppression in neonatal preterm pigs.

Preterm infants are at high risks of sepsis and necrotizing enterocolitis (NEC). Some develop sepsis shortly after suspected or confirmed NEC, implying that NEC may predispose to sepsis but the underlying mechanisms are unknown. Using NEC-sensitive preterm pigs as models, we investigated the immune status in animals following development of subclinical NEC-like lesions with variable severities. Caesarean-delivered preterm pigs were reared until day 5 or day 9. Blood was analyzed for T-cell subsets, neutrophil phagocytosis, transcriptomics, and immune responses to in vitro LPS challenge. Gut tissues were used for histology and cytokine analyses. Pigs with/without macroscopic NEC lesions were scored as healthy, mild, or severe NEC. Overall NEC incidence was similar on day 5 and day 9 (61%-62%) but with lower severity on day 9, implying gradual mucosal repair following the early phase of NEC. Pigs with NEC showed decreased goblet cell density and increased MPO+ and CD3+ cell infiltration in the distal small intestine or colon. Mild or severe NEC lesions had limited effects on circulating parameters on day 5. On day 9, pigs with NEC lesions (especially severe lesions) showed systemic immune suppression, as indicated by elevated Treg frequency, impaired neutrophil phagocytosis, low expression of genes related to innate immunity and Th1 polarization, and diminished LPS-induced immune responses. In conclusion, we shows evidence for NEC-induced systemic immune suppression, even with mild and subclinical NEC lesions. The results help to explain that preterm infants suffering from NEC may show high sensitivity to later secondary infections and sepsis.NEW & NOTEWORTHY Necrotizing enterocolitis (NEC) and sepsis are common diseases in preterm infants. Many develop sepsis following an episode of suspected NEC, suggesting NEC as a predisposing factor for sepsis but mechanisms are unclear. Using preterm pigs as a model, now we show that subclinical NEC lesions, independent of clinical confounding factors, induces systemic immune suppression. The results may help to explain the increased risks of infection and sepsis in preterm infants with previous NEC diagnosis.

Prenatal inflammation suppresses blood Th1 polarization and gene clusters related to cellular energy metabolism in preterm newborns.

Chorioamnionitis (CA, fetal membrane inflammation) predisposes to preterm birth and is associated with increased neonatal infection risk, but the separate effects of prematurity, CA, and postnatal adaptations on this risk are unclear. Using pigs as models for infants, we examined the systemic immune-metabolic status in cesarean-delivered preterm pigs, with and without CA induced by intra-amniotic (IA) LPS exposure. At birth, cord blood of preterm pigs showed neutropenia and low expressions of innate and adaptive immune genes, relative to term pigs. IA LPS induced CA and fetal systemic innate immune activation via complement and neutrophil-related pathways. These were mainly modulated via cellular regulations rather than granulopoiesis, as validated by the in vitro LPS stimulation of cord blood. After birth, IA LPS-exposed preterm pigs did not follow normal immune-metabolic ontogenies found in fetuses or newborns without prenatal insults, but showed consistently high levels of Treg, impaired Th1 polarization, and reduced expressions of multiple genes related to cellular oxidative phosphorylation and ribosomal activities. In conclusion, our results provide cellular and molecular evidence for CA-induced distinct neonatal immune-metabolic status with increased disease tolerance strategy, suggesting mechanisms for the clinical observation of elevated sepsis risks in immune-compromised preterm infants born with CA.

Gut transit time, using radiological contrast imaging, to predict early signs of necrotizing enterocolitis.

BACKGROUND: Immature gut motility in preterm neonates may be a risk factor for necrotizing enterocolitis (NEC). Using preterm pigs as a model for infants, we hypothesized that intestinal dysmotility precedes NEC development. METHODS: Eighty-five preterm pigs were fed increasing amounts of milk diets to induce NEC lesions, as detected at autopsy on day 5. Gut transit time was determined on day 4 by x-ray imaging after oral intake of contrast solution. RESULTS: No clinical or radiological signs of NEC were detected on day 4, but macroscopic NEC lesions were recorded in 59% of pigs (n = 50) on day 5. Relative to pigs without NEC (noNEC, n = 35), pigs with small intestinal lesions (siNEC, n = 18) showed delayed stomach emptying time (StEmpty) and time for contrast to reach cecum (ToCecum) already on day 4. Pigs with lesions only in colon (coNEC, n = 20) showed more diarrhea, shorter ToCecum time, but longer small intestinal emptying time (SiEmpty). ToCecum time predicted siNEC and coNEC lesions with a receiver-operator characteristic area under the curve of 78-81%. CONCLUSIONS: Region-dependent changes in gut transit time is associated with early NEC development in preterm pigs. How gut dysmotility is related to NEC in preterm infants requires further investigations. IMPACT: Using preterm pigs as a model for preterm infants, we show that gut transit time, using serial x-ray contrast imaging, was changed in individuals with NEC-like lesions before they showed the typical radiological signs of NEC. Thus prolonged transit time across the entire gut was recorded when NEC lesions appeared in the small intestine but not when lesions were detected only in the colon. Until now, recordings of food transit have mainly investigated changes in the upper gut. Using serial x-rays, this study describes food transit across the entire gut and documents a region-dependent effect of NEC lesions on gut transit changes in preterm individuals. The findings provide proof of concept for use of x-ray contrast imaging as a tool to monitor gut transit in preterm pigs as models for infants. Delayed passage across the entire gut may be an early sign of small intestinal NEC, at least in pigs. More studies are needed to confirm relations in infants. In the future, it might be possible to use x-ray contrast imaging in preterm infants to better understand gut motility in relation to early NEC progression and need for medical NEC treatment.

Early Use of Antibiotics Is Associated with a Lower Incidence of Necrotizing Enterocolitis in Preterm, Very Low Birth Weight Infants: The NEOMUNE-NeoNutriNet Cohort Study.

OBJECTIVE: To determine whether commencement of antibiotics within 3 postnatal days in preterm, very low birth weight (VLBW; ≤1500 g) infants is associated with the development of necrotizing enterocolitis (NEC). STUDY DESIGN: Preplanned statistical analyses were done to study the association between early antibiotic treatment and later NEC development, using the NEOMUNE-NeoNutriNet cohort of VLBW infants from 13 neonatal intensive care units (NICUs) in 5 continents (n = 2831). NEC incidence was compared between infants who received early antibiotics and those who did not, with statistical adjustments for NICU, gestational age, birth weight, sex, delivery mode, antenatal steroid use, Apgar score, and type and initiation of enteral nutrition. RESULTS: The incidence of NEC was 9.0% in the group of infants who did not receive early antibiotics (n = 269), compared with 3.9% in those who did receive early antibiotics (n = 2562). The incidence remained lower in the early antibiotic group after stepwise statistical adjustments for NICU (OR, 0.57; 95% CI, 0.35-0.94, P < .05) and other potential confounders (OR, 0.25; 95% CI, 0.12-0.47; P < .0001). CONCLUSIONS: In this large international cohort of preterm VLBW infants, a small proportion of infants did not receive antibiotics just after birth, and these infants had a higher incidence of NEC. It is important to better understand the role of such variables as time, type, and duration of antibiotic treatment on NEC incidence, immune development, gut colonization, and antibiotic resistance in the NICU.

Gut and immune effects of bioactive milk factors in preterm pigs exposed to prenatal inflammation.

Prenatal inflammation may predispose to preterm birth and postnatal inflammatory disorders such as necrotizing enterocolitis (NEC). Bioactive milk ingredients may help to support gut maturation in such neonates, but mother's milk is often insufficient after preterm birth. We hypothesized that supplementation with bioactive ingredients from bovine milk [osteopontin (OPN), caseinoglycomacropeptide (CGMP), colostrum (COL)] supports gut, immunity, and NEC resistance in neonates born preterm after gram-negative infection before birth. Using preterm pigs as a model for preterm infants, fetal pigs were given intraamniotic injections of lipopolysaccharide (LPS; 1 mg/fetus) and delivered 3 days later (90% gestation). For 5 days, groups of LPS-exposed pigs were fed formula (FOR), bovine colostrum (COL), or formula enriched with OPN or CGMP. LPS induced intraamniotic inflammation and postnatal systemic inflammation but limited effects on postnatal gut parameters and NEC. Relative to FOR, COL feeding to LPS-exposed pigs showed less diarrhea, NEC severity, reduced gut IL-1ß and IL-8 levels, greater gut goblet cell density and digestive enzyme activities, and blood helper T-cell fraction. CGMP improved neonatal arousal and gut lactase activities and reduced LPS-induced IL-8 secretion in intestinal epithelial cells (IECs) in vitro. Finally, OPN tended to reduce diarrhea and stimulated IEC proliferation in vitro. No effects on villus morphology, circulating cytokines, or colonic microbiota were observed among groups. In conclusion, bioactive milk ingredients exerted only modest effects on gut and systemic immune parameters in preterm pigs exposed to prenatal inflammation. Short-term, prenatal exposure to inflammation may render the gut less sensitive to immune-modulatory milk effects. NEW & NOTEWORTHY Prenatal inflammation is a risk factor for preterm birth and postnatal complications including infections. However, from clinical studies, it is difficult to separate the effects of only prenatal inflammation from preterm birth. Using cesarean-delivered preterm pigs with prenatal inflammation, we documented some beneficial gut effects of bioactive milk diets relative to formula, but prenatal inflammation appeared to decrease the sensitivity of enteral feeding. Special treatments and diets may be required for this neonatal population.

Prenatal Intra-Amniotic Endotoxin Induces Fetal Gut and Lung Immune Responses and Postnatal Systemic Inflammation in Preterm Pigs.

Prenatal inflammation is a major risk for preterm birth and neonatal morbidity, but its effects on postnatal immunity and organ functions remain unclear. Using preterm pigs as a model for preterm infants, we investigated whether prenatal intra-amniotic (IA) inflammation modulates postnatal systemic immune status and organ functions. Preterm pigs exposed to IA lipopolysaccharide (LPS) for 3 days were compared with controls at birth and postnatal day 5 after formula feeding. IA LPS induced mild chorioamnionitis but extensive intra-amniotic inflammation. There were minor systemic effects at birth (increased blood neutrophil counts), but a few days later, prenatal LPS induced delayed neonatal arousal, systemic inflammation (increased blood leukocytes, plasma cytokines, and splenic bacterial counts), altered serum biochemistry (lower albumin and cholesterol and higher iron and glucose values), and increased urinary protein and sodium excretion. In the gut and lungs, IA LPS-induced inflammatory responses were observed mainly at birth (increased LPS, CXCL8, and IL-1ß levels and myeloperoxidase-positive cell density, multiple increases in innate immune gene expressions, and reduced villus heights), but not on postnatal day 5 (except elevated lung CXCL8 and diarrhea symptoms). Finally, IA LPS did not affect postnatal gut brush-border enzymes, hexose absorption, permeability, or sensitivity to necrotizing enterocolitis on day 5. Short-term IA LPS exposure predisposes preterm pigs to postnatal systemic inflammation after acute fetal gut and lung inflammatory responses.

Synbiotics Combined with Glutamine Stimulate Brain Development and the Immune System in Preterm Pigs.

Background: Preterm infants are born with an immature gut, brain, and immune system, predisposing them to short- and long-term complications. Objective: We hypothesized that a milk diet supplemented with pre- and probiotics (i.e. synbiotics) and glutamine would improve gut, brain, and immune maturation in preterm neonates, using preterm pigs as a model. Methods: Preterm pigs (Landrace x Yorkshire x Duroc, n = 40, delivered by c-section at 90% of gestation) were reared individually until day 23 after birth under highly standardized conditions. Piglets in the intervention group (PPG, n = 20) were fed increasing volumes of bovine milk supplemented with prebiotics (short-chain galacto- and long chain fructo-oligosaccharides 9:1, 4-12 g/L), probiotics (Bifidobacterium breve M16-V, 3 × 109 CFU/d) and l-glutamine [0.15-0.30 g/(kg · d)], and compared with pigs fed bovine milk with added placebo compounds as control (CON, n = 20). Clinical, gastrointestinal, immunological, cognitive, and neurological endpoints were measured. Results: The PPG pigs showed more diarrhea but weight gain, body composition, and gut parameters were similar between the groups. Cognitive performance, assessed in a T-maze, was significantly higher in PPG pigs (P < 0.01), whereas motor function and exploratory interest were similar between the groups. Using ex vivo diffusion imaging, the orientation dispersion index in brain cortical gray matter was 50% higher (P = 0.04), and fractional anisotropy value was 7% lower (P = 0.05) in PPG pigs compared with CON pigs, consistent with increased dendritic branching in PPG. In associative fibers, radial diffusivity was lower and fractional anisotropy was higher in PPG pigs compared with CON pigs (all P < 0.05), while measures in the internal capsule showed a tendency towards reduced radial diffusivity and mean diffusivity (both P = 0.09). On day 23 pigs in the PPG group showed higher blood leukocyte numbers (+43%), neutrophil counts (+100%), and phagocytic rates (+24%), relative to CON, all P < 0.05. Conclusion: Preterm pigs supplemented with Bifidobacterium breve, galacto- and fructo-oligosaccharides, and l-glutamine showed enhanced neuronal and immunological development. The findings indicate the potential for targeted nutritional interventions after preterm birth, to support development of important systems such as immunity and brain.

Animal models of chemotherapy-induced mucositis: translational relevance and challenges.

Chemotherapy for cancer patients induces damaging tissue reactions along the epithelium of the gastrointestinal tract (GIT). This chemotherapy-induced mucositis (CIM) is a serious side effect of cytotoxic drugs, and several animal models of CIM have been developed, mainly in rodents and piglets, to help understand the progression of CIM and how to prevent it. Animal models allow highly controlled experimental conditions, detailed organ (e.g., GIT) insights, standardized, clinically relevant treatment regimens, and discovery of new biomarkers. Still, surprisingly few results from animal models have been translated into clinical CIM management and treatments. The results obtained from specific animal models can be difficult to translate to the diverse range of CIM manifestations in patients, which vary according to the antineoplastic drugs, dose, underlying (cancer) disease, and patient characteristics (e.g., age, genetics, and body constitution). Another factor that hinders the direct use of results from animals is inadequate collaboration between basic science and clinical science in relation to CIM. Here, we briefly describe CIM pathophysiology, particularly the basic knowledge that has been obtained from CIM animal models. These model studies have indicated potential new preventive and ameliorating interventions, including supplementation with natural bioactive diets (e.g., milk fractions, colostrum, and plant extracts), nutrients (e.g., polyunsaturated fatty acids, short-chain fatty acids, and glutamine), and growth factor peptides (e.g., transforming growth factor and glucagon-like peptide-2), as well as manipulations of the gut microbiota (e.g., prebiotics, probiotics, and antibiotics). Rodent CIM models allow well-controlled, in-depth studies of animals with or without tumors while pig models more easily make clinically relevant treatment regimens possible. In synergy, animal models of CIM provide the basic physiological understanding and the new ideas for treatment that are required to make competent decisions in clinical practice.

Neonatal gut and immune maturation is determined more by postnatal age than by postconceptional age in moderately preterm pigs.

Preterm infants have immature organ functions that predispose them to gut and immune disorders. Developmental delays at preterm birth may affect various organs differently at term-corrected age. We hypothesized that gut and immune maturation in moderately preterm neonates depends more on birth and postnatal factors than on advancing postconceptional age (PCA). Using preterm pigs as models, we investigated how gut and immune parameters develop until term-corrected age and how these differ from those in term counterparts. Preterm ( n = 43, 106 days of gestation) and term pigs ( n = 41, 116 days of gestation) were delivered by caesarean section and euthanized at birth ( day 1) or postnatal day 11 (term-corrected age for preterm pigs) using identical rearing conditions. Relative to term pigs, preterm pigs had lower blood oxygenation, glucose, and cortisol levels, lower gut lactase activity, villus height, and goblet cell density, and lower blood neutrophil, helper T, and cytotoxic T cell numbers at birth. Despite slower growth in preterm pigs, most intestinal and immune parameters increased markedly after birth in both groups. However, some parameters remained negatively affected by preterm birth until postnatal day 11 (goblet cells, gut permeability, and cytotoxic T cells). The colon microbiota showed limited differences between preterm and term pigs at this time. At the same PCA, preterm 11-day-old pigs had higher blood leukocyte numbers and gut enzyme activities but lower villus height and blood cytotoxic T cell numbers relative to newborn term pigs. Birth and postnatal factors, not advancing PCA, are key determinants of gut and immune maturation in moderately preterm neonates. NEW & NOTEWORTHY Postnatally, preterm infants are often considered to reach a physiological maturation similar to that in term infants when they reach term-corrected postconceptional age (PCA). Using preterm pigs as models, we show that PCA may be a poor measure of gut and immune maturation because environmental triggers (regardless of PCA at birth) are critical. Possibly, PCA is only relevant to evaluate physiological maturation of organs that develop relatively independent of the external environment (e.g., the brain).

Necrotizing enterocolitis is associated with acute brain responses in preterm pigs.

BACKGROUND: Necrotizing enterocolitis (NEC) is an acute gut inflammatory disorder that occurs in preterm infants in the first weeks after birth. Infants surviving NEC often show impaired neurodevelopment. The mechanisms linking NEC lesions with later neurodevelopment are poorly understood but may include proinflammatory signaling in the immature brain. Using preterm pigs as a model for preterm infants, we hypothesized that severe intestinal NEC lesions are associated with acute effects on the developing hippocampus. METHODS: Cesarean-delivered preterm pigs (n = 117) were reared for 8 days and spontaneously developed variable severity of NEC lesions. Neonatal arousal, physical activity, and in vitro neuritogenic effects of cerebrospinal fluid (CSF) were investigated in pigs showing NEC lesions in the colon (Co-NEC) or in the small intestine (Si-NEC). Hippocampal transcriptome analysis and qPCR were used to assess gene expressions and their relation to biological processes, including neuroinflammation, and neural plasticity. Microglia activation was quantified by stereology. The neuritogenic response to selected proteins was investigated in primary cultures of hippocampal neurons. RESULTS: NEC development rapidly reduced the physical activity of pigs, especially when lesions occurred in the small intestine. Si-NEC and Co-NEC were associated with 27 and 12 hippocampal differentially expressed genes (DEGs), respectively. These included genes related to neuroinflammation (i.e., S100A8, S100A9, IL8, IL6, MMP8, SAA, TAGLN2) and hypoxia (i.e., PDK4, IER3, TXNIP, AGER), and they were all upregulated in Si-NEC pigs. Genes related to protection against oxidative stress (HBB, ALAS2) and oligodendrocytes (OPALIN) were downregulated in Si-NEC pigs. CSF collected from NEC pigs promoted neurite outgrowth in vitro, and the S100A9 and S100A8/S100A9 proteins may mediate the neuritogenic effects of NEC-related CSF on hippocampal neurons. NEC lesions did not affect total microglial cell number but markedly increased the proportion of Iba1-positive amoeboid microglial cells. CONCLUSIONS: NEC lesions, especially when present in the small intestine, are associated with changes to hippocampal gene expression that potentially mediate neuroinflammation and disturbed neural circuit formation via enhanced neuronal differentiation. Early brain-protective interventions may be critical for preterm infants affected by intestinal NEC lesions to reduce their later neurological dysfunctions.

Chorioamnionitis, neuroinflammation, and injury: timing is key in the preterm ovine fetus.

BACKGROUND: Antenatal infection (i.e., chorioamnionitis) is an important risk factor for adverse neurodevelopmental outcomes after preterm birth. Destructive and developmental disturbances of the white matter are hallmarks of preterm brain injury. Understanding the temporal effects of antenatal infection in relation to the onset of neurological injury is crucial for the development of neurotherapeutics for preterm infants. However, these dynamics remain unstudied. METHODS: Time-mated ewes were intra-amniotically injected with lipopolysaccharide at 5, 12, or 24 h or 2, 4, 8, or 15 days before preterm delivery at 125 days gestational age (term ~ 150 days). Post mortem analyses for peripheral immune activation, neuroinflammation, and white matter/neuronal injury were performed. Moreover, considering the neuroprotective potential of erythropoietin (EPO) for perinatal brain injury, we evaluated (phosphorylated) EPO receptor (pEPOR) expression in the fetal brain following LPS exposure. RESULTS: Intra-amniotic exposure to this single bolus of LPS resulted in a biphasic systemic IL-6 and IL-8 response. In the developing brain, intra-amniotic LPS exposure induces a persistent microgliosis (IBA-1 immunoreactivity) but a shorter-lived increase in the pro-inflammatory marker COX-2. Cell death (caspase-3 immunoreactivity) was only observed when LPS exposure was greater than 8 days in the white matter, and there was a reduction in the number of (pre) oligodendrocytes (Olig2- and PDGFRα-positive cells) within the white matter at 15 days post LPS exposure only. pEPOR expression displayed a striking biphasic regulation following LPS exposure which may help explain contradicting results among clinical trials that tested EPO for the prevention of preterm brain injury. CONCLUSION: We provide increased understanding of the spatiotemporal pathophysiological changes in the preterm brain following intra-amniotic inflammation which may aid development of new interventions or implement interventions more effectively to prevent perinatal brain damage.

Nutrient Fortification of Human Donor Milk Affects Intestinal Function and Protein Metabolism in Preterm Pigs.

Background: Nutrient fortification of human milk is often required to secure adequate growth and organ development for very preterm infants. There is concern that formula-based fortifiers (FFs) induce intestinal dysfunction, feeding intolerance, and necrotizing enterocolitis (NEC). Bovine colostrum (BC) may be an alternative nutrient fortifier, considering its high content of protein and milk bioactive factors. Objective: We investigated whether BC was superior to an FF product based on processed bovine milk and vegetable oil to fortify donor human milk (DHM) for preterm pigs, used as a model for infants. Methods: Sixty preterm pigs from 4 sows (Danish Landrace × Large White × Duroc, birth weight 944 ± 29 g) received decreasing volumes of parenteral nutrition (96-72 mL â‹… kg-1 â‹… d-1) and increasing volumes of enteral nutrition (24-132 mL â‹… kg-1 â‹… d-1) for 8 d. Pigs were fed donor porcine milk (DPM) and DHM with or without FF or BC fortification (+4.6 g protein â‹… kg-1 â‹… d-1). Results: DPM-fed pigs showed higher growth (10-fold), protein synthesis (+15-30%), villus heights, lactase and peptidase activities (+30%), and reduced intestinal cytokines (-50%) relative to DHM pigs (all P < 0.05). Fortification increased protein synthesis (+20-30%), but with higher weight gain and lower urea and cortisol concentrations for DHM+BC compared with DHM+FF pigs (2- to 3-fold differences, all P ≤ 0.06). DHM+FF pigs showed more diarrhea and reduced lactase and peptidase activities, hexose uptake, and villus heights relative to DHM+BC or DHM pigs (30-90% differences, P < 0.05). Fortification did not affect NEC incidence but DHM+BC pigs had lower colonic interleukin (IL)-6 and IL-8 concentrations relative to the remaining pigs (-30%, P = 0.06). DHM+FF pigs had higher stomach bacterial load than did DHM, and higher bacterial density along intestinal villi than did DHM and DHM+BC pigs (2- to 3-fold, P < 0.05). Conclusions: The FF product investigated in this study reduced growth, intestinal function, and protein utilization in DHM-fed preterm pigs, relative to BC as fortifier. The relevance of BC as an alternative nutrient fortifier for preterm infants should be tested.

Growth and Clinical Variables in Nitrogen-Restricted Piglets Fed an Adjusted Essential Amino Acid Mix: Effects of Partially Intact Protein-Based Diets.

Background: Current recommendations for protein levels in infant formula are intended to ensure that growth matches or exceeds growth of breastfed infants, but may provide a surplus of amino acids (AAs). Recent infant studies with AA-based formulas support specific adjustment of the essential amino acid (EAA) composition allowing for potential lowering of total protein levels. With the use of a combination of intact protein and free EAAs, we designed a formula that meets these adjusted EAA requirements for infants. Objective: Our objective was to test whether this adjusted formula is safe and supports growth in a protein-restricted piglet model, and whether it shows better growth than an isonitrogenous formula based on free AAs. Methods: Term piglets (Landrace × Yorkshire × Duroc, n = 72) were fed 1 of 4 isoenergetic formulas containing 70% intact protein and 30% of an EAA mixture or a complete AA-based control for 20 d: standard formula (ST-100), ST-100 with 25% reduction in proteinaceous nitrogen (ST-75), ST-75 with an adjusted EAA composition (O-75), or a diet as O-75, given as a complete AA-based diet (O-75AA). Results: After an initial adaptation period, ST-75 and O-75 pigs showed similar growth rates, both lower than ST-100 pigs (∼25 compared with 31 g · kg-1 · d-1, respectively). The O-75AA pigs showed further reduced growth rate (15 g · kg-1 · d-1) and fat proportion (both P < 0.05, relative to O-75). Conclusions: Formula based partly on intact protein is superior to AA-based formula in this experimental setting. The 25% lower, but EAA-adjusted, partially intact protein-based formula resulted in similar weight gain with a concomitant increased AA catabolism, compared with the standard 25% lower standard formula in artificially reared, protein-restricted piglets. Further studies should investigate if and how the specific EAA adjustments that allow for lowering of total protein levels will affect growth and body composition development in formula-fed infants.

Growth and Clinical Variables in Nitrogen-Restricted Piglets Fed an Adjusted Essential Amino Acid Mix: Effects of Free Amino Acid-Based Diets.

Background: Excess protein intake in early life has been linked to obesity and metabolic syndrome in later life. Yet protein, and in particular the essential amino acids (EAAs), need to be present in adequate quantity to support growth. Objective: With the use of a piglet model restricted in dietary amino acids (AAs), we compared the efficacy and safety of a standard formula with a low-AA formula containing an adjusted AA composition. Methods: Female piglets (3-7 d old; Landrace × Yorkshire × Duroc) were fed 1 of 4 isoenergetic AA-based formulas for 14 d (700 kJ · kg body weight-1 · d-1). The formulas contained a set control amount (44 g/L) and AA compositions referred to as the experimental standard (ST-100, n = 22), or 20% or 50% lower total AAs (respectively, ST-80, n = 19 and ST-50, n = 13), or 20% lower total AAs with an optimally adjusted EAA composition (O-80, n = 17). A series of clinical and paraclinical endpoints were measured. Results: Growth rates were similar for ST-100, O-80 and ST-80 piglets (all ∼15 g · kg-1 · d-1), whereas ST-50 had a markedly lower weight gain relative to all groups (all P < 0.05). Relative to ST-100, all groups with reduced AA intake showed ∼16% reduction in plasma albumin and ∼30% reduction in plasma urea (both P < 0.05). The absolute leucine oxidation rate was ∼30% lower for O-80 than for ST-100 piglets (P < 0.05). Conclusions: These data show that a 20% reduction in total AA intake for both the control (ST-80) and the adjusted AA (O-80) formula did not have any short-term adverse effects on growth in artificially reared, AA-restricted piglets. The lower absolute leucine oxidation rate observed in O-80 supports the development of an infant formula with an improved AA composition and a moderate reduction in total protein to support adequate growth in healthy infants.

Bioactive Whey Protein Concentrate and Lactose Stimulate Gut Function in Formula-fed Preterm Pigs.

OBJECTIVE: Formula feeding is associated with compromised intestinal health in preterm neonates compared with maternal milk, but the mechanisms behind this are unclear. We hypothesized that the use of maltodextrin and whey protein concentrates (WPCs) with reduced bioactivity owing to thermal processing are important factors. METHOD: Ninety-two cesarean-delivered preterm pigs were fed increasing doses of formulas for 5 days (24-120 mL ·â€Škg ·â€Šday). In experiment 1, 4 groups of pigs (n = 15-16) were fed lactose- or maltodextrin-dominant formulas (lactose/maltodextrin ratios 3:1 or 1:3, respectively), containing WPC with either high or low levels of IgG (WPC1 or WPC2, respectively). In experiment 2, 2 groups of pigs (n = 15-16) were fed lactose-dominant formulas with either a bioactive WPC (BioWPC, produced by reduced thermal-processing) or a conventional WPC (ConWPC). RESULTS: In experiment 1, pigs fed formula with WPC1 had higher villi, hexose absorption, and lactase activity in small intestine, relative to WPC2, but predominantly with the lactose-dominant formula (all P < 0.05). In experiment 2, the BioWPC product had higher bioactivity, as indicated by higher IgG, lactoferrin, and TGF-ß2 levels, and better enterocyte proliferation in vitro. Pigs fed the BioWPC formula showed better feeding tolerance and higher intestinal villi and lactase activity (all P < 0.05). The BioWPC formula-fed pigs also had greater physical activity (P < 0.05 on day 4) and tended to show improved hexose absorption and decreased gut permeability (both P ≤ 0.09). CONCLUSIONS: Infant formulas containing lactose as the main carbohydrate, and WPC with reduced thermal processing, may support gut maturation and health in sensitive, preterm neonates.

Chemotherapeutic treatment reduces circulating levels of surfactant protein-D in children with acute lymphoblastic leukemia.

BACKGROUND: Surfactant protein D (SP-D) is a host defense molecule of the innate immune system that enhances pathogen clearance and modulates inflammatory responses. We hypothesized that circulating SP-D levels are associated with chemotherapy-induced mucositis and infectious morbidity in children with acute lymphoblastic leukemia (ALL). PROCEDURE: In a prospective study, 43 children receiving treatment for ALL were monitored for mucosal toxicity from diagnosis through the induction phase of treatment. Serial blood draws were taken to determine the levels of SP-D, interleukin-6 (IL-6), C-reactive protein, and white blood cells. Data on fever, antibiotics, and bacteremia were collected. Baseline levels of circulating SP-D were compared with healthy controls. RESULTS: Baseline values of circulating SP-D were similar to levels in healthy controls (median: 829 ng/ml vs. 657 ng/ml, respectively, P > 0.05). After initiation of chemotherapy, a significant reduction in SP-D levels was observed at all time points: 704 ng/ml at day 8, 413 ng/ml at day 15, 395 ng/ml at day 22, and 520 ng/ml at day 29 (all, P < 0.05). No significant associations between SP-D values, the occurrence of mucosal toxicity, or infectious morbidity were observed. However, loss of circulating SP-D from days 8 to 15 was associated with more systemic inflammation, and lower SP-D values at day 15 were associated with elevated intestinal mucositis scores (P < 0.05). CONCLUSIONS: The current study supports the hypothesis that the detrimental effect of chemotherapy on patients' immune functions includes decreased circulating levels of innate mucosal molecules such as SP-D, potentially aggravating mucosal and systemic inflammatory responses.